Journal cf Applied Bacteriology 1989,67, 363-370 292611 0188

Lactobacillus plantarum; a deleterious contaminant of plant

tissue cultures

c. L E I F E R T * t f , W . M . WAIITES?, H E L E NC A M O T T A ? & J . R .
N I C H O L A?Department
S~~ of Applied Biochemistry and Food Science, University of
Nottinyham School of Agriculture, Sutton Bonington, Louyhborough, L E l 2 5RD, U K and
tNeoPlants Ltd., Freckleton, t’R4 I H U , Lancashire, U K

Received 20 October 1988, revised 15 February 1989 and accepted 17 March 1989

L E I F E R TC., , . M . , C A M O T T AH, . & N I C H O L A SJ.R.

, W A I T E SW , 1989. Lacto-
hacillus plantarum; a deleterious contaminant of plant tissue cultures. Journal of
Applied Bacteriology 67, 363-370.
Lactohacillus plantarum was isolated from in vitro plant cultures o f Hemerocallis
Stella d o r a , Catherine Woodhury and Stafford. Infected cultures deteriorated
rapidly during three subcultures (15 weeks) when grown in uitro, showing chlorotic
white shoots and grey calli. Weaned plants developed normally when transferred to
the soil. The drop in multiplication rate of plant cultures coincided with a decrease
in pH of the growth media. Uninfecied plants of Hemerocallis Stella d’ora showed
the same symptoms after inoculation with L. plantarum. Lactohacillus plantarum was
reisolated from inoculated plant cultures that showed symptoms, thus fulfilling
Koch’s postulates. In plants inoculated with L. plantarum both the amount of DL-
lactic acid formed and the number of plants showing symptoms increased with
increasing numbers of bacteria in the inoculum, wheras plant multiplication rate
and pH decreased. These effects could be reproduced by adding ur-lactic acid to the
multiplication medium of plants free From L. planturum, suggesting that the bacterial
production of lactic acid was responsible for the changes in infected plants.

Micropropagation of plants is usually defined as
the aseptic multiplication of plant calli or shoots
on defined media in uitro. Division of plant
material and subculture on fresh media every 4
weeks can result in multiplication rates between
two and ten per subculture.
Although aseptic condition!; are usually
implied, many plant cultures are not or d o not
stay aseptic in vitro (Boxus & Terzi 1987;
Singha et nl. 1987). Of the various contaminants
of micropropagated plants bacteria are con-
sidered the most serious (Cornu & Michel 1987,
Leggat et nl. 1988). Few of the bacteria isolated
from micropropagated plants have been
described sufficiently to allow identification to
species level (Knauss & Miller 1978; Trick &
* Corresponding author.
Lingens 1985; Leifert et al. 1989). Of the species
described only Erwinia carotouora is a ‘classic’
plant pathogen (Knauss & Miller 1978). Most of
the other species described are not known to be
pathogenic to plants in uivo, but are saprophytes
in soil, on plants or in human tissues (Leifert et
al. 1989).
T o our knowledge no attempts have been
made to establish the mechanism by which non-
pathogens can harm plants in v i m . Decrease in
yield of potatoes due to non-pathogens has been
linked in viuo to the production of bacterial
metabolites or toxins such as cyanide (Bakker
& Schippers 1987, Schippers et a/ . 1987), but
similar mechanisms have still to be established
for in uitro grown plants.
We have tried to overcome this gap in our
understanding of plant/bacteria relationships in
 C. Leifert et al.
vitro in this study by establishing the pathogenic
nature of one particularly important contami-
nant, Lactobacillus plantarum, and studying the
changes in the composition of plant growth
media in the presence of infected cultures.
Material and Methods
P L A N T MATERIAL, P L A N T G R O W T H MEDIA
AND GROWTH CONDITIONS
Hemerocallis Stella d’ora (Liliaceae) plants were
used. Plants were multiplied on the basic
mineral medium of Murashige & Skoog (1962)
containing (mg/l) indole-3-acetic acid (IAA), 0.2;
6-(y,y-dimethylallylamino) purine (2iP), 25
(medium MS19). Ten plant pieces were planted
per tub and incubated in a growth room at
20°C in the light and 18°C in the dark. Only
uninfected or L. plantarum infected plants were
used and assessed in the experiments. Cross-
contaminated tubs were discarded.
DETERMINATION OF MULTIPLICATION
RATE A N D MEDIUM pH
Ten callus pieces (0.5-1.0 cm diameter with 2-3
green shoots) were planted in each tub with
fresh medium. The multiplication rate was
determined by assessing the number of callus
pieces with similar size per tub after 4 weeks in
the growth room. The pH of the plant medium
was measured directly in the medium with a
Corning 220 pH meter after the plants had been
grown for 4 weeks. The medium pH was also
determined before and after autoclaving and
after 4 weeks in the growth room without
plants.
I N F E C T I O N T E S T I N G OF P L A N T M A T E R I A L
Shoot, callus and agar pieces from each tub
used for the trials described were aseptically
transferred into a 25 ml screw-capped bottle
with 10 ml of sterility test medium [half strength
Murashige & Skoog (1962) medium altered as
described by Leifert et al. (1989)l at every sub-
culture to confirm their sterility. Turbidity
developing in the medium indicated the pre-
sence of an infected plant.
ISOLATION A N D IDENTIFICATION OF
BACTERIA
Bacteria were either isolated directly from
growth on the plant multiplication media or
from turbid sterility test medium and then
transferred to nutrient-broth yeast extract agar
(NBY; Schaad 1980).
Bacteria from each contaminated tub were
identified as L . plantarum by their growth char-
acteristics on sterility test medium and Hugh &
Leifson’s (1953) medium, anaerobic growth on
De Man, Rogosa and Sharp’s agar (MRS; De
Man et al. 1960), catalase test (MacFaddin
1980) and Gram stain (Jensen’s modification).
Fifteen strains were further studied with the
API 20B and 50 CHL systems (API-bio-
Merieux, Basingstoke, UK) and the results
obtained analysed by the API computer identifi-
cation service (Leifert et al. 1989). During the
experiment, bacteria isolated from turbid steril-
ity test medium were examined for anaerobic
growth on MRS and for Gram stain reaction,
catalase production and microscopic appear-
ance to confirm infection with L. plantarum. For
reinoculation of uninfected plant cultures we
used L. plantarum strain 016, which had been
isolated previously from a tub of Hemerocallis
Stella d’ora plants showing symptoms. Strains
reisolated from reinoculated plant cultures were
identified as L. plantarum strain 016 by catalase
test, Gram stain, anaerobic growth on MRS
agar and API 50 CHL results.
D E T E R M I N A T I O N O F G R O W T H OF
Lactobacillus plantarum
Screw-capped, 25 ml bottles containing 10 ml of
liquid plant multiplication medium MS19
(composition see above) or MS19 enriched with
MRS-broth but without plants were inoculated
with a 48 h MRS culture of L. plantarum to give
a final turbidity of 0.1 (f0.02) determined at
625 nm using a Grating spectrophotometer
(Cecil Instruments). Growth of L. plantarum in
medium MS19 with or without MRS-broth was
again determined by measuring E,,, in the
medium.
INOCULATION WITH Lactobacillus plantarum
Plants were dipped into a bacterial suspension
(E,,, of 1.0 diluted tenfold in Ringer’s solution)
of a 48 h anaerobic MRS culture of strain 0/6,
 L. plantarum in plunt tissue cultures 365
Table 1. Media composition and bacterial inocula
~~

Inoculum of
Lactobacillus Plants
Sucrose Lactic acid plantarum Per
Medium code (gp) (gP) (cfu,’ml) treatment
1 (control) 40 0 0 450

B4 40 0 10’ 50
B3 40 0 103 50
B2 40 0 1O6 50
B1 40 0 108 50

39.95 0.053 0 50
39.9 0.105 0 50
39.15 0.264 0 50
39.5 0.523 0 50
37 3,158 0 50

before subculturing to new media (Table 1).
Uninoculated control plants were dipped into
sterile Ringer’s solution. From each initial tub
10 plants were used for reinoculation and 10
plants as controls. Control and contaminated
plants were subcultured into separate tubs.
Colony counts on MRS agar were used to esti-
mate the number of colony forming units (cfu)
in the inoculum. For the reisolation trial 200
callus pieces (20 tubs) were inoculated with
2 x 10’ cfu. Fifty callus pieces (5 tubs) were
used for all other inocula investigated.

I
n 4

10 15 20 25
First Tirne after first detection of L.p/unlurum (weeks)
plants
showing
symptoms
Fig. 1. Multiplication rate and medium p H of Hemerocallis Stella d’ora cultures either free or infected with
Lactobacillus plantarum. 0, multiplication rate of uninfected plants; 0 , multiplication rate of Lactobacillus plan-
tarum infected plants; 0, medium p H of uninfected plants; B, medium pH of Lactobacillus plantarum infected
plants. Mean is of 10 p H determinations.
366 C. Leifert et al.
Fig. 2. Symptoms of Lactobacillus plantarurn infected Hemerocallis Stella d’ora plants. Left tub: infected plants.
Right tub: non-infected plants.
EXTRACTION A N D DETERMINATION OF
D(-) AND L(+) LACTIC ACID
The amount of D(-)- and lactic acid in
multiplication medium MS19 was determined 4
weeks after the plants had been inoculated with
L. plantarum. The agar medium (20 g) from each
tub was homogenized in 40 ml distilled water,
left in a waterbath at 80°C for 15 min and cen-
trifuged at 3100 rev/min for 10 min before the
supernatant fluid was used for determination of
lactic acid. The amount of D(-)- and L( +)-
lactic acid in the supernatant fluid was assayed
with a L-lactic acid kit and lactate dehy-
drogenase (Boeringer).
ADDITION OF LACTIC ACID TO P L A N T
M U L T I P L I C A T I O N M E D I U M MS19
Sucrose was substituted in plant multiplication
medium MS19 by molar equivalents of DL-lactic
acid (free acid 98%, 85% syrup; Sigma). Lactic
acid was filter-sterilized and added to the multi-
plication medium after autoclaving (Table 1).
Fifty plants ( 5 tubs) were used for each concen-
tration of DL-lactic acid.
Results
PERFORMANCE OF PLANTS INFECTED
WITH Lactobacillus plantarum
Multiplication rate and medium pH of infected
Hemerocallis Stella d‘ora plants began to
decrease 10 weeks after L. plantarum had been
detected as a contaminant (Fig. 1). Within 15
weeks all infected plants were dead, showing
chlorotic white shoots and grey calli (Fig. 2).
Uninfected plants continued to multiply nor-
mally, the pH in the medium stayed between 4.7
and 5 and no plants with symptoms could be
observed. Cultures of Hemerocallis Stafford and
Catherine Woodbury infected with L . plantarum
deteriorated in a similar manner, showing the
same symptons and fall in medium pH to below
3.5.
R E I S O L A T I O N OF Lactobacillus plantarum AND
P E R F O R M A N C E OF I N F E C T E D C U L T U R E S
When L. plantarum strain 016 was inoculated on
uninfected Hemerocallis Stella d’ora plants,
Gram-negative, catalase-positive rods growing
 L. plantarum in plant tissue cultures 367
Table 2. Reisolation results and performance of plants free and infected with Lactobacillus plantarurn
Lactobacillus Lactobacillus
plantarum free plantarum infected
n X F n X S

Reisolation of Lactobacillus plantarum (%) 17 0 0 19 100 0

Plants with symptoms (%) 17 0 0 19 99.5 0.22
Multiplication rate 17 22 0 19 0.12 0.10
Medium pH 17 4.99 0.15 19 3.24 0.06
n = Number of tubs (10 plants/tub) assessed.
X = Mean.
s = Standard Deviation.

anaerobically on MRS medium could be iso-
lated from all tubs containing inoculated plants
after 4 weeks. All strains tested had the same
API 50 CHL profile as L. planturum strain 0/6.
No other bacterium could be isolated from
these tubs, nor could L. plantarum be isolated
from control plants. Ninety-nine per cent of the
L. plantarum infected plants showed the symp-
toms seen in Fig. 2 and the multiplication rate
dropped to below 0.2 and the ptI to an average
of 3.2 within 4 weeks after inoculation. Control
plants appeared healthy, multiplied normally
and did not show any drop in pH (Table 2).
G R O W T H OF Lactobacillus plantarum O N
PLANT GROWTH MEDIUM WITHOUT
PLANTS
No growth of L. plantarum in medium MS19
without plants could be detected by the
methods applied (the turbidity in the medium
did not increase above 0.1). Growth of L. plan-
tarum could be induced by adding MRS-broth
to medium MS19. Addition of 1 g/1 resulted in
an increase of turbidity to about 0.5 and 10 g/l
resulted in a turbidity of about 0.9.
RE-INOCULATION O F UNINFECTED P L A N T S
WITH Lactobacillus plantarum
When uninfected plants were inoculated with L.
plantarum and grown for 4 weeks on MS19 the
multiplication rate and medium pH decreased
(Fig. 3), whereas the m-lactic acid content in
the medium and the number of plants showing
symptoms increased (Fig. 4). A bacterial inocu-
lum of lo6 to 10' cfu/ml resulted in the pro-
duction of about 1.5 g m-lactic acid/kg of
medium, a medium pH of below 3.5,with more
than half the plants dead and all of them

5ti L

I
I 1 I I 1

I 10 lo3 I o6 lo8
lnoculum ( c f u / m l )
Fig. 3. Multiplication rate and medium pH of plant cultures inoculated with Lactobacillus plantarum. 0,
multi-
plication rate of plants; 0,
medium pH. Mean is of 5 pH determinations.
368 C. Leifert et al.
0-
1-1 I I
0 10 I o3
lnoculum
Fig. 4. D(+)- and L(-)-lactic acid concentration in plant growth media and percentage of plants showing symp-
toms 4 weeks after inoculation with Lactobacillus plantarurn. 0, D( +)-lactic acid; m, L( -)-lactic acid; 0,
lactic acid; x , percentage of plants showing symptom. Mean is of two lactic acid determinations.
showing the symptoms described above. A low
bacterial inoculum of 10 cfu/ml did not cause a
significant decrease in multiplication rate after 4
weeks, although L. plantarum established itself
on the plant and the production of 0.1 g/kg DL-
lactic acid in the medium could be detected. In
addition the pH fell to below 4, and 17% of
plants showed the symptoms described. The
amount of m-lactic acid in media from un-
infected plants was below the amounts detect-
able by the method used.
PERFORMANCE OF PLANTS GROWN ON
MEDIUM ~ s 1 C9O N T A I N I N G LACTIC ACID
Multiplication rate and medium pH decreased
and the number of plants showing symptoms
increased with increasing concentrations of
lactic acid in the plant growth medium (Fig. 5).
Addition of lactic acid at concentrations of 0.05,
0.1 and 0.25 mg/l did not reduce the multiplica-
tion rate of plants significantly and only addi-
tion of 0.25 mg/l or more lactic acid resulted in
plants showing symptoms. A dramatic drop in
multiplication rate was observed when lactic
acid was added at 0.5 g/l when more than 50%
of the plants lost viability and showed the
typical symptoms. Addition of 3 g/1 DL-lactic
acid resulted in only dead plants.
I I I 1 I
Io6 I o8
(cfu/ml)
total
I N F L U E N C E OF T H E P L A N T O N pH OF THE
MEDIUM
The pH of medium containing lactic acid and
inoculated with plants is significantly higher
than that of medium kept in the growth room
for 4 weeks with lactic acid and without plants
(Fig. 5).
Discussion
Lactobacilli are acidophilic bacteria which can
grow at pH values below four (Kandler & Weiss
1986). Lactobacillus plantarum is facultatively
heterofermentative and ferments glucose homo-
fermentatively to DL-lactic acid, but five carbon
sugars are fermented heterofermentative to DL-
lactic acid and acetic acid (Schlegel 1981). Acid
production by L. plantarum has long been used
to preserve human and animal food such as
sauerkraut, salami or silage (Schlegel 1981).
In this study we have shown that L. plan-
tarum is pathogenic for micropropagated Hem-
erocallis plants. The rate of growth of L.
plantarum on plant growth medium appears to
be increased by plant exudates as growth could
not be detected on plant growth medium in the
absence of the plant. Further evidence for this
view arises from the observation that visible
growth of L. plantarurn in plant cultures is
 L. plantarum in plant tissue cultures
5

I
a

I I I I I I 1
0 0.5 I 2 3
Lactic acid ( 9 1 1 )

Fig. 5. Multiplication rate and medium pH after plants were grown for 4 weeks on medium containing lactic
acid. 0, multiplication rate of plants; x , percentage of plants showing symptoms; 0, medium pH. Medium
without plants was also incubated for 4 weeks after addition of lactic acid and the pH (H)
determined. Mean is of
five pH determinations.

restricted t o the zones close t o the base of the
plant.
Growth of L. plantarum was shown to
produce lactic acid in the agar medium and this
was found to be responsible for the drop in
multiplication rate and deterioration of plant
cultures. A direct toxicity of lactic acid rather
than the associated p H drop as the cause for
plant decay is further indicated by the finding
that plant cultures infected with fungi with a
medium pH of between 2.5 to 3.0 show no
apparent symptoms.
The amount of lactic acid produced and the
extent to which plant growth was affected was
shown to depend on the level of 1 he initial bac-
terial inoculum. Suppression of the bacterium
for limited periods in culture with antibiotics or
other bactericidal or bacteriostatic agents
might be expected to allow plant growth.
Weaning of symptomless plants also stops
plants from dying, probably because L. plan-
tarum is not very competitive in oivo. We have
been able to eliminate completely L. plantarum
from plant cultures using antibiotics
(unpublished results).
Toxicity of lactic acid in concentrations
above 0.1 g/kg produced by L. plantarum or
above 0.25 g/kg when incorporated into the
medium of uninfected plants could indicate that
production of lactic acid is not the only mecha-
nism by which L. plantarum harms the plant.
Another explanation for the different quantities
needed to induce symptoms could be that lactic
acid produced by L. plantarum is not evenly dis-
tributed throughout the medium, but concen-
trated around the plant base where most of the
bacterial growth occurs. Further investigations
into the distribution of lactic acid and possibly
other bacterial metabolites need to be carried
out to answer these questions.
Toxicity of lactic acid in concentrations of
about 0.1 to 0.3 g/kg in the medium indicates
that lactic acid bacteria in general should be
considered a serious threat to the successful
micropropagation of plants.
The skilful assistance of Mr Kevin Ashworth is
gratefully acknowledged.
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